1. Field of the Invention
The present invention relates generally to the fields of molecular hematology and protein chemistry. More specifically, the present invention relates to a novel treatment for sickle cell anemia.
2. Description of the Related Art
Hemoglobinopathies encompass a number of anemias of genetic origin in which there is decreased production and/or increased destruction (hemolysis) of red blood cells. The blood of normal adult humans contains hemoglobin (designated as HbA) which contains two pairs of polypeptide chains designated alpha and beta. Fetal hemoglobin (HbF), which produces normal red blood cells, is present at birth, but the proportion of HbF decreases during the first months of life and the blood of a normal adult contains only about 2% HbF. There are genetic defects which result in the production by the body of abnormal hemoglobins with a concomitant impaired ability to maintain oxygen concentration. Among these genetically derived anemias are included thalassemia, Cooley's Disease and, most importantly, sickle-cell anemia (HbS disease).
Sickle-cell anemia is an inherited chronic hemolytic anemia characterized by sickle-shaped red blood cells present in part of the offspring of parents who are both heterozygotes to the abnormal gene which causes the sickling disease. This disease is recessive, and heterozygotes carrying this gene show no blatant anemia or similar abnormality. Thus, only about 25% of the children of parents who are both heterozygous are expected to be homozygotic to this abnormal gene and will develop sickle cell anemia and eventually sickling crisis (aplastic crisis). Few homozygotes live past 40 years of age and many show abnormal body growth patterns. The gene which characterizes sickling trait causes valine to be substituted for glutamic acid in the sixth position of the beta chain, thus producing HbS rather than HbA. Deoxygenated HbS is much less soluble than deoxy HbA and it forms a semisolid gel of rodlike tactoids, thus causing the red blood cells produced from HbS to assume a sickle shape. These abnormally shaped red blood cells form a sort of sludge. In addition, these HbS red blood cells are more fragile than normal red blood cells and hemolyze more easily, thus leading eventually to anemia. The clinical manifestations of an aplastic crisis in sickle-cell homozygotes include arthralgia with fever, jaundice, aseptic necrosis of the femoral head, chronic punched-out ulcers about the ankles plus episodes of severe abdominal pain with vomiting. Thrombosis and/or infarction may also be present. Laboratory findings include a monocytic anemia with an RBC count in the range 2-3 times. Early death, usually before 40, is caused by intercurrent infections (especially tuberculosis), multiple pulmonary emboli or thrombosis of a vessel supplying a vital area. In the past, treatment of sickle-cell anemia was symptomatic only.
Recently, however, it has been found that drugs which can increase production of the normal fetal hemoglobin HbF (since clearly, drugs cannot alter the HbS/HbA ratio in homozygotes since it is genetically determined), can tide a homozygote over the aplastic crisis, and thus potentially prolong their life. It has been known for some time that drugs such as 5-azacytidine, cytarabine and hydroxyurea could augment HbF production in anemic monkeys--see Levine et al, New Eng. J. Med. 310:869 (1984). Recent limited clinical studies have shown that these drugs do indeed increase HbF production in patients with sickle-cell disease--see Goldberg et al, New Eng. J. Med. 323:366 (1990) for hydroxyurea; Characheet al., Blood 69:109 (1988); 6th Annual Conf. on Hemoglobin Switching, Sep. 2, 1988 for 5-azacytidine and hydroxyurea; Veith et al, New Eng. J. Med. 313:1571 (1985) for cytarabine and hydroxyurea. In addition to the previously cited experiments in anemic monkeys (Levin et al loc. cit.), more recently Constantoulakis et al, Blood 77:1326 (1991) have developed a new model system for studying the induction of fetal hemoglobin (HbF) by various drugs, using adult transgenic mice carrying the human A (gamma) globin gene linked to the locus control region regulatory sequences and expressing heterocellularly HbF. Erythropoietin, 5-azacytidine, hydroxyurea and butyric acid esters (butyrate), all known in vivo HbF inducers in adult humans, also induced HbF in this model. Further, large scale human trials with hydroxyurea have been conducted. (Chavache et al., N. Engl. J. Med., 332:1317-1322 (1995).
The molecular events which occur within red blood cells from homozygous sickle cell (SS) patients and to their extracellular environment leading to the painful sickle cell crisis, organ damage, and mortality are of great interest to the clinical and scientific community (reviews Hebbel, 1990, 1991, Powers, 1990, Francis, Jr. and Johnson, 1991, Joiner, 1993). Blood from SS patients can be separated on density gradients into morphologically and physiologically distinct red blood cell classes (Fabry et al, 1984). During the course of vaso-occlusion the highest density class of red blood cells are selectively trapped in the microvasculature (Kaul et al, 1986, 1989). This high density class of red blood cells include irreversibly sickled cells (ISCs) (60-85%) that retain a sickled shape in well oxygenated blood, and unsickleable SS dense discocytes (USDs) (Kaul et al, 1983). These observations explain why ISCs and USDs are reduced in the peripheral blood during a sickle cell crisis (Fabry et al, 1984, Ballas et al, 1988, Lande et al, 1988, Ballas and Smith, 1992). The ISCs appear to block the narrowed lumen of vessels lined primarily with the more adherent lower density reversibly sickled cells (RSCs), and sometimes by direct capillary occlusion (Kaul et al, 1989, Fabry et al, 1992).
Twenty years ago, Lux and coworkers made the observation that most red blood cell membranes (ghosts) isolated from ISCs remain sickled, and triton skeletons prepared from ISC ghosts all remain sickled (Lux et al, 1976). These observations demonstrated that after removal of all of the hemoglobin (HbS) from the ISC RBC, and most of the membrane phospholipids and integral membrane proteins, the remaining skeleton retained the sickled shape. When sickled RSC's were triton-extracted the resulting skeletons did not retain their sickled shape. In order for the released skeletons to remodel their shape, protein associations between spectrin, protein 4.1, and actin protofilaments (and other accessory proteins) must be dissociated, and then new interactions formed.
The red blood cell contains a two dimensional latticework of fibrous proteins which covers the cytoplasmic surface of its plasma membrane. This supramolecular structure, termed the membrane skeleton, maintains the biconcave shape of the erythrocyte, gives it essential properties of elasticity and flexibility for its circulatory travels, controls the lateral mobility of integral membrane proteins, and serves as a structural support for the bilayer (review, Goodman et al, 1988). The essential core components of this two dimensional meshwork are spectrin, f-actin, and protein 4.1 (Yu et al, 1973, Sheetz, 1979), although triton membrane skeletons isolated at moderate ionic strength conditions (such as those utilized by Lux et al (1976)) contain other more minor components.
Erythrocyte spectrin is primarily an (.alpha..beta.).sub.2 tetrameric flexible rod of 200 nm extended contour length, formed by head-to-head linkage of two .alpha..beta. heterodimers (Shotton et al, 1979). Cloning and cDNA sequencing of both the .alpha. subunit (Sahr et al, 1990) and .beta. subunit (Winkelmann et al, 1990) have indicated molecular weights of 280 kD (.alpha.) and 246 kD (.beta.) for the spectrin subunits. Essential to the formation of the two dimensional membrane skeleton is the ability of spectrin tetramers to bind actin filaments at both ends, thereby crosslinking f-actin (Brenner and Korn, 1979, Cohen et al, 1980, Shen et al, 1986). The actin binding domain of human RBC spectrin has been localized to a stretch of 140 amino acids at the N terminus of .beta. spectrin from alanine.sup.47 through lysine.sup.186 (Karinch et al, 1990). Erythrocyte actin protofilaments observed on electron microscopy of negatively stained intact membrane skeletons fall within a narrow range of lengths, with a mean length of 33 to 37 nm in control (AA) red blood cells, equivalent to a double-stranded helix with 14 actin monomers (Shen et al, 1986, Byers and Branton, 1985). The extended skeleton appears to be primarily a hexagonal lattice (Liu et al, 1987) with actin protofilaments (and associated proteins) at the center and six corners of the hexagons, interconnected by spectrin tetramers (.about.85%) and three armed hexamers (.about.10%). The spectrin-actin interaction is strengthened by a peripheral membrane protein, protein 4.1, which also binds to the ends of the spectrin tetramers (Tyler et al, 1979, Ungewickell et al, 1979, Fowler and Taylor, 1980). Therefore spectrin, actin protofilaments, and protein 4.1 constitute the core RBC skeleton.
Other accessory proteins to the skeleton include protein 4.9 which bundles f-actin in vitro (Siegel and Branton 1985), tropomyosin which lines the grooves of actin protofilaments (Fowler and Bennett, 1984), and adducin a Ca.sup.2+ -calmodulin binding protein which stimulates the addition of spectrin to f-actin in a protein 4.1-independent manner (Gardner and Bennett, 1987, Mische et al, 1987). The spectrin membrane skeleton is attached to the membrane by at least two types of interactions. Ankyrin binds to .beta. spectrin 20 nm from the junction of the heterodimers and also binds to the integral membrane protein band 3 (Bennett and Stenbuck 1979, 1980, Yu and Goodman, 1979, Hargreaves et al, 1980, Wallin et al, 1984). The second membrane linkage is based on the ability of protein 4.1 to bind to an integral membrane protein (Shiffer and Goodman, 1984) which appears to be glycophorin C (Mueller and Morrison, 1981).
Previous attempts to look at membrane skeletal defects within the sickle cell have focussed on the membrane linkage proteins. Platt et al (1985) demonstrated that SS spectrin depleted inside-out vesicles (IOVs) bound .about.50% less spectrin in vitro than did control AA IOV. While this suggested a potential ankyrin defect, purified ISC ankyrin bound spectrin normally in vitro. Schwartz et al (1987) demonstrated that SS protein 4.1 was more aggregated upon isolation than AA protein 4.1, and bound protein 4.1-depleted IOVs less effectively than AA IOV's. While both of these studies point to potentially important alterations in the linkage between the core skeleton and the SS bilayer, neither could explain the persistently sickled membrane skeleton observed on triton X-100 extraction of ISC ghosts (Lux et al, 1976). In the triton extracted skeletons the bilayer has been removed, yet the ISC skeleton remained sickled.
Hebbel et al (1982) have demonstrated that sickle cells generate about twice the amount of activated oxygen species found in normal red blood cells. The basis for this increase in oxygen radicals is the combined result of accelerated autoxidation of HbS to methemoglobin, a conversion which causes a release of heme (Hebbel et al, 1988). Heme is increased in content on the cytoplasmic surface of sickle cell membranes, and this increase correlates with the amount of membrane protein thiol modification (Kuross et al, 1988). It is therefore not surprising that spectrin, band 3, ankyrin, and protein 4.1 all have some degree of thiol modification (Rank et al, 1985, Schwartz et al, 1987). While the thiol modifications of spectrin and ankyrin are reversible with DTT (Rank et al, 1985), the oxidation of thiols in protein 4.1 is not reversible (Schwartz et al, 1987). Schwartz et al (1987) have reported that SS protein 4.1 contains 1-2 mole % fewer cysteine than control protein 4.1, and 1 mole % cysteic acid not found in control protein 4.1.
The prior art is deficient in the lack of effective means of treating sickle cell anemia. The present invention fulfills this longstanding need and desire in the art.